P612: ASSESSMENT OF THE DEVELOPMENT OF B CELL MALIGNANCIES IN A NOVEL MURINE MODEL CARRYING DLEU2 DELETION
Authors: Alice Ramassone, Shimaa Hassan Abdelaziz Soliman, Enrica Placidi, Pasquale Rea, Carola DI Camillo, Vincenzo Coppola, Rosa Visone
Journal: HemaSphere (2023)

Background: The 13q14 deletion is the most frequent genetic abnormality in Chronic Lymphocytic Leukemia (CLL) and it is one of the causes of the downregulation of miR-15a and miR-16-1, which contributes to CLL development. The 13q14 deletion has been identified also in other B-cells malignancy, such as Monoclonal B-cell lymphocytosis, Diffuse Large B Cell Lymphoma, and Multiple Myeloma. In CLL, the 13q14 deletion shows heterogeneity in length, the minimal deleted region contains a partial sequence of the Deleted in lymphocytic Leukemia 2 gene (DLEU2), which is the host gene of miR-15a/16-1 cluster, but it does not always include the miRs. However, miR-15a/16-1 cluster is transcribed as a part of a long DLEU2 transcript and deletion of DLEU2 promoter also results in down regulation of these miRs. In CLL patients carrying the 13q14 deletion, the miR-15a/16-1 cluster shows a transcriptional regulation mediated by the RNA polymerase III (RPIII), which correlates with the poor prognostic factor ZAP70. Nevertheless, the impact on CLL progression is not yet clear. Aims: To elucidate whether and how the miRs-transcriptional regulation mediated by RPIII impacts on the CLL development and progression. Methods: We generated two novels murine models carrying two different deletions of the Dleu2 gene: i) dleu2ex1-4 lacking the promoter and the first four exons of murine Dleu2, but not miR-15a/16-1 locus, thus making the locus available only to the RPIII; ii) dleu2ex5-6 lacking the exons five and six of murine Dleu2, but not miR-15a/16-1 locus, thus retaining the canonical RPII regulation. We crossed floxed mice with EIIA-CRE and CD19-CRE mice, to get the deletion both in germline and in tissue-specific condition. We monitored the emergency of a B-cells malignancies over time by FACS by analysing the percentage of B220+ and B220+CD5+ cells in peripheral blood from mice at 6 and 12 months of age. We evaluated the presence of a B-cell malignancies by FACS and IHC in tissues collected from mice sacrificed at 18 months of age. We analysed the expression of Dleu2 and miR-15a in purified B cells using RT-qPCR; we evaluated the presence of an RPII-independent transcript by 5’- and 3’- Rapid Amplification of cDNA Ends (RACE). Results: We sacrificed 32 mice at 18 months of age, having both germline and tissue-specific Dleu2 deletions; among them, 10 were WT, 2 were deleted at dleu2ex1-4, and 20 were deleted at dleu2ex5-6. Overall, in mice carrying Dleu2 deletions, we detected lymphoproliferative disorders: one mouse (ex1-4loxP/+) showed the accumulation of B220+CD5+ cells in the spleen, while another one (ex5-6+/-) developed a B cell lymphoma diffused in the spleen, lymph node, liver, and pancreas; furthermore, a fraction of mice both WT (1 of 5) and with germline deletion at dleu2ex5-6 (4 of 13) developed the amyloidosis. Regarding the involvement of the RPIII in the transcription of miR-15a/16-1 locus in the novel mouse models, we found that in B cells collected from a mouse with dleu2ex1-4 deletion miR-15a expression is independent of Dleu2 expression; accordingly, we identified an alternative transcript at miR-15a/16-1 locus using 3’- and 5’- RACE, suggesting that an alternative promoter lies between the miR-15a/16-1 locus and the exon 4 of Dleu2. Summary/Conclusion: Despite the small sample number, our preliminary data suggest that both Dleu2 deletions could promote the development of a lymphoproliferative disorder in aged mice; however, up to now, the small number of mice did not allow us to define whether the RPIII regulation impact differently on the development and progression of a B cells malignancies. Keywords: Transcriptional regulation, B cell chronic lymphocytic leukemia, Animal model, B cell lymphoma